Malolactic enzyme from Oenococcus oeni: Heterologous expression in Escherichia coli and biochemical characterization

Malolactic enzymes (MLE) are known to directly convert L-malic acid into L-lactic acid with a catalytical requirement of nicotinamide adenine dinucleotide (NAD(+)) and Mn(2+); however, the reaction mechanism is still unclear. To study a MLE, the structural gene from Oenococcus oeni strain DSM 20255 was heterologously expressed in Escherichia coli, yielding 22.9 kU l(−1) fermentation broth. After affinity chromatography and removal of apparently inactive protein by precipitation, purified recombinant MLE had a specific activity of 280 U mg(−1) protein with a recovery of approximately 61%. The enzyme appears to be a homodimer with a molecular mass of 128 kDa consisting of two 64 kDa subunits. Characterization of the recombinant enzyme showed optimum activity at pH 6.0 and 45°C, and K(m), V(max) and k(cat) values of 4.9 mM, 427 U mg(−1) and 456 sec(−1) for L-malic acid, 91.4 µM, 295 U mg(−1) and 315 sec(−1) for NAD(+) and 4.6 µM, 229 U mg(−1) and 244 sec(−1) for Mn(2+), respectively. The recombinant MLE retained 95% of its activity after 3 mo at room temperature and 7 mo at 4°C. When using pyruvic acid as substrate, the enzyme showed the conversion of pyruvic acid with detectable L-lactate dehydrogenase (L-LDH) activity and oxidation of NADH. This interesting observation might explain that MLE catalyzes a redox reaction and hence, the requirements for NAD(+) and Mn(2+) during the conversion of L-malic to L-lactic acid.